Prevention of tumors with monoclonal antibodies against neu

ABSTRACT

Methods of preventing the transformation of a normal cell into a tumor cell that has p185 on its surface are disclosed. The methods comprise administering an antibody which specifically binds to p185. Methods of preventing the transformation of a normal cell into a tumor cell that has p185 on its surface in an individual at high risk of developing tumors are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 08/525,800, which is a371 of PCT/US94/03528, filed Mar. 30, 1994, which is acontinuation-in-part of Ser. No. 08/038,498, filed Mar. 30, 1993,abandoned.

FIELD OF THE INVENTION

The invention relates to methods of preventing the transformation ofnormal mammalian cells into tumor cells.

BACKGROUND OF THE INVENTION

Huge amounts of time and money have been spent to better understandcancer and searching for ways to prevent and cure cancer. The results ofthese research efforts have provided a greater understanding of thebiological and biochemical events that participate in the formation oftumors.

Tumor cells display a variety of characteristics that distinguish themfrom normal cells. Recent studies in the molecular genetics of cancerindicate that certain genes known as oncogenes may play a role in thetransformation of some cells from their normal condition to a cancerouscondition.

An oncogene which encodes a protein that exposes antigenic sites on thesurface of transformed cells has been identified by transfection of DNAfrom ethyl nitrosourea-induced rat neuroblastomas into NIH3T3 cells.This oncogene has been termed neu. The neu gene has been found to beamplified in some human tumors, particularly those of the breast,suggesting that this gene may play a role in the etiology of humancancer.

The neu oncogene encodes a cell surface protein on rat cells transformedby it. The protein encoded by the neu oncogene is a 185 kDatransmembrane glycoprotein with tyrosine kinase activity, generallyknown by the name p185. The neu gene is closely related to the epidermalgrowth factor (EGF) receptor gene in structure.

The neu oncogene and p185 have also been found active in humanadenocarcinomas including breast, lung, salivary gland and kidneyadenocarcinomas, as well as prostate neuroblastoma. In human primarybreast cancers, amplification of the neu oncogene was found in about 30%of all malignant tumors examined. Increased stage of malignancy,characterized by large tumor size and increased number of positive lymphnodes as well as reduced survival time and decreased time to relapse,was directly correlated with an increased level of amplification of theneu gene. The neu protooncogene is expressed at low levels in normalhuman tissues. Further, neu has been associated with 100% of the ductalcarcinomas studied in situ, Lodato, R. F., et al. (1990) Modern Pathol.3(4):449.

While changes in diet and behavior can reduce the likelihood ofdeveloping cancer, it has been found that some individuals have a higherrisk of developing cancer than others. Further, those individuals whohave already developed cancer and who have been effectively treated facea risk of relapse and recurrence.

Advancements in the understanding of genetics and developments intechnology as well as epidemiology allow for the determination ofprobability and risk assessment an individual has for developing cancer.Using family health histories and/or genetic screening, it is possibleto estimate the probability that a particular individual has fordeveloping certain types of cancer. Those individuals that have beenidentified as being predisposed to developing a particular form ofcancer can take only limited prophylactic steps towards reducing therisk of cancer. There is no currently available method or compositionwhich can chemically intervene with the development of cancer and reducethe probability a high risk individual will develop cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer or are otherwise in remission areparticularly susceptible to relapse and reoccurrence.

There is a need for improved preventative agents for individual with ahigh risk to develop cancer and for individuals who have had cancerenter remission or be removed. In cases where the type of cancer theindividual is at risk to develop, such as tumors associated with neu,there is a need for specific agents which can be administered to reducethe probability that a predisposed individual will develop cancer orthat a patient in remission will suffer a relapse.

SUMMARY OF THE INVENTION

The present invention provides methods for the prevention of tumor cellswhich express a translation product of the neu oncogene on theirsurfaces. In accordance with the invention, a prophylactic amount of anantibody that specifically binds to p185 is administered to anindividual.

The present invention provides methods of preventing the transformationof normal human cells into tumors cells which express a translationproduct of the neu oncogene on their surfaces. In accordance with theinvention, a prophylactic amount of an antibody that specifically bindsto p185 is administered to an individual.

The present invention provides methods for the prevention of theorigination of genetically induced mammalian tumor cells which express atranslation product of the neu oncogene on their surfaces by interferingwith a transforming event. In accordance with the invention, aprophylactic amount of an antibody that specifically binds to p185 isadministered to an individual.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “neu-associated cancer” and “neu-associatedtumors” are meant to refer to tumor cells and neoplasms which expressthe neu gene to produce p185.

The translation product of the neu oncogene is p185, a transmembraneglycoprotein having tyrosine kinase activity and a molecular weight ofabout 185,000 daltons as determined by carrying out electrophoresis onthe glycoprotein and comparing its movement with marker proteins ofknown molecular weight. Experiments have shown that administration of anantibody binding to p185 results in the reduced incidence ofneu-associated tumors in a population susceptible to such tumors.Anti-p185 antibodies selectively inhibit the neoplastic development inanimals susceptible to developing neu transformed tumors.

The occurrence of mammalian tumors cells which express a translationproduct of the neu oncogene on their surfaces can be prevented byadministration of antibodies which bind to p185. In accordance with theinvention, a prophylactic amount of an antibody that specifically bindsto p185 is administered to an individual who is identified as beingsusceptible to neu-associated tumors.

The present invention is particularly useful to prophylactically treatan individual who is predisposed to develop neu-associated tumors or whohas had neu-associated tumors and is therefore susceptible to a relapseor recurrence.

As used herein, the term “high risk individual” is meant to refer to anindividual who has had a neu-associated tumor either removed or enterremission and who is therefore susceptible to a relapse or recurrence.As part of a treatment regimen for a high risk individual, theindividual can be prophylactically treated against the neu-associatedtumors that they have been diagnosed as having had in order to combat arecurrence. Thus, once it is known that an individual has had cancercharacterized by tumor cells with p185 on their cell surfaces, theindividual can be treated according to the present invention to preventnormal cells from transforming into tumor cells.

Prophylactic compositions for prevention of neu-associated tumorscomprise an antibody specific for the p185 molecule and apharmaceutically acceptable carrier. According to preferred embodiments,the prophylactic compositions for prevention of neu-associated tumorsare injectable. The compositions comprise an antibody specific for thep185 molecule and a pharmaceutically acceptable carrier or injectionvehicle.

The antibodies are chosen from antibodies made according to theprocedures described in detail below or other conventional methods forproducing monoclonal antibodies. The carrier be selected from those wellknown to persons having ordinary skill in the art. An example of acarrier is sterile saline.

Antibodies Specific for Rat and Human p185

Those having ordinary skill in the art can produce monoclonal antibodieswhich specifically bind to p185 and are useful in prophylacticanti-tumor compositions using standard techniques and readily availablestarting materials. The techniques for producing monoclonal antibodiesare outlined in Harlow, E. and D. Lane, (1988) ANTIBODIES: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., which isincorporated herein by reference, provide detailed guidance for theproduction of hybridomas and monoclonal antibodies which specificallybind to target proteins.

Briefly, the protein of interest, rodent or human p185 for example, isinjected into mice. The spleen of the mouse is removed, the spleen cellsare isolated and fused with immortalized mouse cells. The hybrid cells,or hybridomas, are cultured and those cells which secrete antibodies areselected. The antibodies are analyzed and, if found to specifically bindto the protein of interest, the hybridoma which produces them iscultured to produce a continuous supply of antigen specific antibodies.

According to the present invention, antibodies specific for eitherrodent, particularly rat, p185 or the corresponding human p185 may beused in prophylactic compositions. Accordingly, either rodent p185 orhuman p185 is used to generate hybridomas. In both cases, the geneswhich encode these proteins are widely known and readily available tothose having ordinary skill in the art. Thus, one having ordinary skillin the art can make antibodies useful to practice the present invention.In addition to rodent antibodies, the present invention relates to humanantibodies, humanized antibodies, Fabs and chimeric antibodies and Fabswhich bind to p185 which may be produced routinely by those havingordinary skill in the art.

In some preferred embodiments of the present invention, the prophylacticcomposition comprises monoclonal antibodies designated 7.5.5, 7.9.5,7.16.4 and 7.21.2. In some preferred embodiments of the presentinvention, the prophylactic composition comprises humanized monoclonalantibodies or Fabs which contain complementarity determining regionsfrom antibodies designated 7.5.5, 7.9.5, 7.16.4 and 7.21.2. In somepreferred embodiments of the present invention, the prophylacticcomposition comprises humanized monoclonal antibodies or Fabs whichcontain variable regions from antibodies designated 7.5.5, 7.9.5, 7.16.4and 7.21.2.

Patient Population

Although the present invention may be used to prevent tumors in anypatient population identified as being susceptible to neu-associatedtumors, it is particularly useful in high risk individuals who, forexample, have a family history of neu-associated cancer or show agenetic predisposition. Additionally, the present invention isparticularly useful to prevent neu-associated tumors in patients whohave had neu-associated tumors removed by surgical resection or who havebeen diagnosed as having neu-associated cancer in remission.

Those having ordinary skill in the art can readily identify individualswho are susceptible to neu-associated tumors, particularly thoseindividuals considered to be a high risk for whom the methods of theinvention are particularly useful.

Compositions

The prophylactic compositions may include additional components torender them more effective. For example, a prophylactic composition ofthe invention may comprise multiple anti-p185 antibodies includingantibodies specific for different epitopes of p185.

The prophylactic compositions may include other anti-cancer agents suchas, for example, cis-platin. As a step in the method of the invention,chemotherapeutics may be administered prophylactically to patients whohave treated for neu-associated cancer by surgery or radiation treatmentand who have had removal or remission.

Administration Regimen

About 5 μg to 5000 mg of antibody may be administered. In some preferredembodiments, 50 μg to 500 mg of antibody may be administered. IN otherpreferred embodiments, 500 μg to 50 mg of antibody may be administered.In a preferred embodiment, 5 mg of antibody is administered.

Prophylactic compositions may be administered by an appropriate routesuch as, for example, by oral, intranasal, intramuscular,intraperitoneal or subcutaneous administration. In some embodiments,intravenous administration is preferred.

Subsequent to initial administration, individuals may be boosted byreadministration. In some preferred embodiments, multipleadministrations are performed.

EXAMPLES Example 1 Mice

C3H and [C3H×DBA/2] F1 (C3D2 F1) mice were obtained from the JacksonLaboratory, Bar Harbor, Me. Inbred congenitally athymic Balb/c nude(nu/nu) mice were obtained from the National Cancer Institute animalcolony (San Diego, Calif.). Animals used in the experiments aremaintained in accordance with the guidelines of the Committee on Careand Use of Laboratory Animals of the Institute of Animal Resources,National Research Council (DHEW publication number (NIH) 78-23, revised1978).

Isolation of Hybridomas that Secrete Monoclonal Antibodies that areReactive with Neu-Transformed Cells

C3H/HeJ mice are repeatedly immunized with NIH 3T3 transfectantstransformed by the neu oncogene (cell line B104-1-1), emulsified inFreund's adjuvant. Spleens from immune mice are fused with theaminopterin-sensitive NS-1 myeloma line, and hybridomas are selected inhypoxanthine-aminopterin-thymidine media. Culture supernatants fromgrowing hybridomas are initially screened for the presence of antibodycapable of binding B104-2-1 cells by indirect immunofluorescence usingfluorescence activated cell sorting (FACS). Positive supernatants arethen tested for specificity by determining whether they contain antibodycapable of binding normal NIH 3T3 cells, or NIH 3T3 cells transformed bytransfection with Harvey sarcoma virus proviral DNA (cell lineXHT-1-1a).

Isotype Analysis of Monoclonal Antibodies

The heavy chain isotypes of the monoclonal antibodies characterized hereare determined by double immunodiffusion in agar according to the methodof Ouchterlony, in Hudson, L and F. C. Hay, eds., Practical Immunology,Blackwell Scientific Publications, London, p. 117, which is specificallyincorporated herein.

Purification of Monoclonal Antibodies

Hybridoma cells are washed several times in HBSS and injected intopristine primed, 400 rad irradiated, C3D2F1 mice to induce ascites fluidproduction. When the mice develop significant ascites, the fluid isremoved by aspiration with a 19 gauge needle and hybridoma cells anddebris are removed by centrifugation at 1000×g. The clarified ascitesfluid is then stored at −70° C. prior to purification, or is purifiedimmediately. Purification is performed according to the method of Drebinet al. in Immunology and Cancer (N. L. Kripke and P. Frost, eds.)University of Texas Press, Austin, Tex., p. 277 which is specificallyincorporated herein.

Specificity of Antibodies Flow Cytometry

Cells are removed from dishes with buffered EDTA (Versene; Gibco) andwashed twice in FACS medium (Hank's balanced salt solution (HBBS; Gibco)supplemented with 2% fetal calf serum (FCS), 0.1% sodium azide and 0.1mM HEPES); 1×10⁶ cells in 0.1 ml FACS medium are incubated with 0.1 mlof hybridoma culture supernatant for 1 hr at 4° C. Cells are washedtwice with FACS medium, and incubated with 0.1 ml fluoresceinisothiocyanate (FITC)-conjugated rabbit-anti-mouse immunoglobulin(Miles) diluted 1:50 in FACS medium for 1 hr at 4° C. Cells are thenwashed twice in FACS medium and fixed in 2%paraformaldehyde-phosphate-buffered saline (PBS). Samples are run on anOrtho 2150 Cytofluorograph using the logarithmic amplifier. Each samplecontains 10,000 cells per sample.

Cyanogen Bromide Coupling of Antibodies to Sepharose Beads

CNBr-activated Sepharose 4B beads are swollen in 1 mM HCl, and thenmixed with purified antibodies in coupling buffer (0.5 M NaCl, 0.1 MNaHC₃, pH 8.3) at a ratio of 2 mg immunoglobulin (1 mg per ml) per ml ofactivated beads. The mixture is rotated overnight on an end-over-endmixture at 4° C., and then unreacted sites are blocked with 0.2 Nglycine pH 8.0 for 2 hours at room temperature. The beads are thenpoured onto a sintered glass filter and washed with three cycles of 100bead volumes of coupling buffer, 10 bead volumes of 3.5 N MgCl₂, 100bead volumes of coupling buffer to wash away excess adsorbed proteins.Non-specific protein binding to the antibody coupled beads is blocked bya brief wash in sterile DMEN containing 10% fetal calf serum. The beadsare then washed in PBS and stored in PBS containing 0.1% sodium azide at4° C. until they are used in immunoprecipitation experiments. All of themonoclonal antibodies which specifically bind to the surface ofneu-transformed cells are reactive with the p-185 molecule encoded bythe neu oncogene. These monoclonal antibodies specifically precipitatep185 from metabolically labeled lysates of neu-transformed cells.

Immunoprecipitation of p185 from Metabolically Labeled b104-1-1 CellLysates

For labeling with ³⁵S-cysteine 16⁶ cells are seeded in 100 mm culturedishes and labelled for 18 hr in 2 ml minimal essential medium (MEM)containing 0.1 the usual amount of cysteine, 2% dialyzed fetal calfserum and 500 μCi ³⁵S-cysteine (77 Ci mmol⁻¹; NEN). For labeling with³²P, 3×10⁵ cells are seeded in 60-mm tissues culture dishes andincubated for 18 hr in 0.8 ml phosphate-free Dulbecco-Vogt modifiedEagle's medium containing 4% fetal calf serum and 0.4 mCi ³²P(carrier-free; NEN). Cells are lysed in phosphate-buffered RIPA buffercontaining 1 mM ATP, 2 mM EDTA and 20 mN sodium fluoride, andimmunoprecipitates are prepared and washed according to Sefton et al.(1979) Virology 28:957-971 (1979), which is specifically incorporatedherein. One third of each lysate is incubated with 1 μl of normal mouseserum or 60× concentrated 7.16.4 culture supernatant at 4° C. for 60min. Sheep anti-mouse immunoglobulin (1 μl; Cappel) is added to eachsample and incubation continued for 30 min. Immune complexes arepelleted using fixed Protein A-bearing Staphylococcus aureus and washed.Samples are analyzed by SDS-polyacrylamide gel electrophoresis in 7.5%acrylamide-0.17% bis-acrylamide gels. The gels are treated forfluorography and exposed to preflashed Kodak X-Omat AR film for 10 days.

Antibodies Specific for Human Neu Oncogene

Rat and human neu oncogene DNA sequences are similar and the two genesshare some sequences as can be shown by computer-aided analysis of thestructure of the genes. Antibodies to the human gene can be produced byfollowing the procedure as set forth above for making antibodies to therat neu oncogene and using the rat neu oncogene sequences which areshared with human neu oncogene instead of the rat neu oncogene.

As a result of competitive binding studies, antibody 7.16.4 was found tobind to domain 1, antibodies 7.5.5, 7.9.5 and All were found to bind todomain 2, and antibody 7.21.2 was found to bind to domain 3. Thedenominations of domains 1, 2, and 3 are arbitrary and are used as ashort hand to group antibodies that competitively bind to p185 into thesame group. Antibodies placed into any one group competitively bind withother antibodies of the same group to p185, but do not to anysubstantial extent inhibit binding of antibodies to other portions ofp185.

Isotype analysis of the antibodies provided the following isotypes forthe antibodies: IgG1-antibody 7.9.5; IgG2a-antibodies A11 and 7.16.4;IgG2B-antibody 7.5.5; and IgG1-antibody 7.21.2.

Hybridoma cell line producing monoclonal antibody 7.9.5 was deposited inthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md., 20852-1776 on Jul. 3, 1990 and has accession number HB10492.Hybridoma cell line producing monoclonal antibody 7.16.4 was depositedin the American Type Culture Collection 12301 Parklawn Drive, Rockville,Md., 20852-1776 on Jul. 3, 1990 and has accession number HB10493.

Example 2

Oncogenic rat neu (neuT) differs from wild type neu by a point mutationwithin the transmembrane domain of the coding sequence. Certain strainsof transgenic mice that express the neuT oncogene (L. Bouchard, et al.Cell S7, 931 (1989)) develop breast tumors at an average of forty fourweeks of age. Intraperitoneal injection of a monoclonal antibody againstp185^(neuT) dramatically affected tumor development in these transgenicmice. A significant proportion (50%) of mice did not develop tumors evenafter ninety weeks of age when injected with monoclonal antibodies. Thisdemonstrates for the first time that immunological manipulations ofp185^(neuT) can effectively prevent the development of geneticallyinduced breast tumors in a rodent model.

In the transgenic mouse models of human breast adenocarcinomas developedby L. Bouchard et al. (Cell 57, 931 (1989) and by W. J. Muller et al.(Cell 54, 105 (1988)), the neuT oncogene under the transcriptionalcontrol of the murine mammary tumor virus (MMTV) long terminal repeatleads to mammary tumors.

In one of these models, female transgenic mice developed multiplemammary adenocarcinomas asynchronously, between 20 and 45 weeks of age(comparable to human middle age) in a stochastic manner. The histologicfeatures and metastatic potential of these adenocarcinomas resembledtumors seen in humans. This transgenic mouse model has certain importantcharacteristics; 1) the expressed oncogene is genetically programmed andis activated in a predictable manner in conjunction with tissue specificpromoter/enhancer elements; 2) the stochastic appearance of tumorssuggests that involvement of other oncogenes or oncogenic factors isnecessary for full development of tumors, a situation clearly analogousto naturally occurring tumors; and 3) the immunological interactionsbetween the tumors and the host transgenic animal can be examined, sincethe host immune system was intact. Finally, the effect of distincttreatments could be assessed on tumors prior to or after their predicteddevelopment.

In the present set of experiments we have employed only female mice ofline MN-10 on the BALB/c background. These mice became pregnantfrequently and were able to nurse their litters during the treatmentperiod.

To determine the effects of MAb specific for the ectodomain ofp185^(neuT) on the development of breast tumors, two groups oftransgenic mice with different dosages of antibodies starting at 6 weeksof age were treated. One group of transgenic mice was injectedintraperitoneally with 10 μg of MAb 7.16.4 in 100 μl of phosphatebuffered saline (PBS) biweekly (low dose group). Another group oftransgenic mice was injected with the same amount of MAb 7.16.4 twiceweekly (high dose group). Each group of mice had comparable numbers ofcontrol transgenic mice treated with injections of PBS only. An isotypematched MAb (IgG2a) known to have no effect on p185^(neuT) a transformedcells in vitro or in vivo was used as a control.

As expected, two groups of control transgenic mice (n=12 and n=10)developed tumors between 28.0 and 72.0 weeks of age. The average tumoronset periods of these two sets of control mice were 42.8±3.2 (SEM) and45.0±3.0 weeks, respectively. The group of mice receiving low doses ofMAb 7.16.4 (n=11) developed tumors between 31.0 and 75.0 weeks of agewith the average tumor onset period 50.7±2.7 weeks. Although the averagetumor onset period was significantly delayed by 7.9 weeks between thelow dosage group and its associated control mice (p<0.05), thedifference between the low dosage group and the second control mice (5.7weeks delay) was marginally insignificant (0.05≦p≦0.1). There was nosignificant difference between the two controls (0.1<p).

The high dose treatment group of mice developed tumors after 45.6 weeksof age. However, 6 of 12 mice in this group (50%) remained free oftumors at more than 90 weeks of age. This indicates that treatment oftransgenic mice with MAb 7.16.4 10 μg twice weekly can effectivelysuppress tumor development in a large fraction of these mice for almosttheir entire life span (about 100 weeks). Nearly half of the mice inboth control groups and in the low dosage group developed two to fiveindependent tumors within a six week period after the first tumor becamevisible. In contrast, all animals that developed malignancy in the highdosage group had only a single tumor. The tumor volume of the highdosage group at a given point after tumor appearance was always smallerthan that of control mice at the same point.

The histology of the MNTV/neuT transgenic nice used in the presentexperiment have been previously characterized in detail (L. Bouchard etal. Cell 57, 931 (1989)) which is incorporated herein by reference. Alluntreated mice developed moderately to poorly differentiated ductaladenocarcinomas of the breast. A small proportion of these mice alsodeveloped salivary gland and Harderian gland tumors consistent withprevious observations. The breast tumors which arose in mice treatedwith MAb 7.16.4 were moderately to poorly differentiated adenocarcinomas(FIGS. 2A and B) which were histologically indistinguishable from thatof untreated mice. Occasionally single tumors displayed both poorly andmoderately differentiated areas. The non-tumor breast tissue of the highdosage treated mice was histologically similar to that of the untreatedmice, even after 70 weeks of treatment No ductal epithelial hyperplasia,ductal destruction or lymphoid infiltration was observed. There is noindication that the suppression of tumor development in MAb treated miceinvolves host immune mechanisms such as antibody-dependent cellularcytotoxicity (ADCC). Similarly, studies using tumor implant model of MAbtherapy found no decisive contribution of host immune elements to theelimination of established tumors expressing neuT.

About 30% of human breast tumors show p185^(c-erB-2) overexpression,usually associated with gene amplification, and it is relevant that theoverexpression of p185^(c-erB-2) can be observed in early stages ofhuman breast tumors. The data indicates that continuous down-regulationof the p185^(neuT) molecule leads to tumor growth suppression in adose-dependent manner. The antibody mediated dose-dependent tumorsuppression shown here suggests that the continuous down-regulation ofp185^(neu)T diminishes the activity of necessary oncogenic factors intumorigenesis. Prevention of metastasis or recurrence is feasible byadministering anti-p185 antibodies.

1. A method of preventing transformation of a normal cell into a tumorcell in an individual at risk of developing a tumor having tumor cellswhich have p185 on their surfaces, said method comprising the steps of:a) identifying said individual; and, b) administering to said individualan antibody which specifically binds to p185.
 2. The method of claim 1wherein the antibody has the complementarity determining regions from anantibody selected from the group consisting of 7.16.4 and 7.9.5.
 3. Themethod of claim 1 wherein the antibody has the variable region from anantibody selected from the group consisting of 7.16.4 and 7.9.5.
 4. Themethod of claim 1 wherein the antibody is selected from the groupconsisting of 7.16.4 and 7.9.5.
 5. The method of claim 1 wherein theantibody is 7.16.4.
 6. The method of claim 1 wherein the antibody is ahumanized antibody with complementarity determining regions selectedfrom an antibody the group consisting of 7.16.4 and 7.9.5.
 7. The methodof claim 1 wherein the antibody is a humanized antibody withcomplementarity determining regions from antibody 7.16.4.
 8. The methodof claim 1 wherein the antibody is a humanized antibody with variableregions selected from an antibody the group consisting of 7.16.4 and7.9.5.
 9. The method of claim 1 wherein the antibody is a humanizedantibody with variable regions from antibody 7.16.4.
 10. The method ofclaim 1 further comprising administering to said individual a secondantibody which specifically binds to p185.
 11. The method of claim 1further comprising administering to said individual an anti-tumor agent.12. A method of preventing transformation of a normal cell into a tumorcell that has p185 on its surface in individual who has had a tumor thathas p185 on its cell surfaces removed or who has had cancercharacterized by tumor cells that have p185 on their surfaces enterremission comprising the steps of: a) identifying said individual; and,b) administering to said individual an antibody which specifically bindsto p185.
 13. The method of claim 12 wherein the antibody has thecomplementarity determining regions from an antibody selected from thegroup consisting of 7.16.4 and 7.9.5.
 14. The method of claim 12 whereinthe antibody has the variable region from an antibody selected from thegroup consisting of 7.16.4 and 7.9.5.
 15. The method of claim 12 whereinthe antibody is selected from the group consisting of 7.16.4 and 7.9.5.16. The method of claim 12 wherein the antibody is 7.16.4.
 17. Themethod of claim 12 wherein the antibody is a humanized antibody withcomplementarity determining regions selected from an antibody the groupconsisting of 7.16.4 and 7.9.5.
 18. The method of claim 12 wherein theantibody is a humanized antibody with complementarity determiningregions from antibody 7.16.4.
 19. The method of claim 12 wherein theantibody is a humanized antibody with variable regions selected from anantibody the group consisting of 7.16.4 and 7.9.5.
 20. The method ofclaim 12 wherein the antibody is a humanized antibody with variableregions from antibody 7.16.4.
 21. The method of claim 12 furthercomprising administering to said individual a second antibody whichspecifically binds to p185.
 22. The method of claim 12 furthercomprising administering to said individual an anti-tumor agent.